Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: RT-PCR of human and murine megakaryocytes and platelets. (A) Murine RT-PCR studies of total RNA from (1) megakaryocyte, (2) platelets, (3) WBCs, (4) NIH-3T3 cells, known to express LRP1,20 and (5) water for LRP1 (first gel), PF4 (second gel), and ITGB2 (third gel). Expected band size is approximately 400 base pair (bp) for LRP1 and ITGB2, and approximately 300 bp for PF4. White space indicates where lanes were removed for ease of presentation. (B) Human RT-PCR studies of total RNA from (6) megakaryocytes, (7) platelets, (8) WBCs, (9) 293T cells, embryonic kidney line known to express LRP1,21 (10) water, and (11) no reverse transcriptase controls for LRP1 (first gel), PF4 (second gel), and ITGB2 (third gel).

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: Flow cytometry and Western blot of human and murine megakaryocytes and platelets. (A) Representative examples of flow cytometry of murine bone marrow–derived megakaryocytes (top) stained with a biotin labeled anti-hLRP1 antibody known to cross-react with mouse LRP131 and then stained with streptavidin, PE–Alexa 647 secondary antibody. The gray line represents unstained cells. The broken black line represents secondary antibody alone. The solid black line is megakaryocytes with both antibodies. The bottom graph shows flow cytometry of platelets similarly performed. (B) As in panel A but for human cultured megakaryocytes and human peripheral blood platelets. LRP1 antibody was directly labeled with Alexa 647 for these experiments. As in panel A, the solid gray line represents unstained cells. The broken black line represents cells with isotype control antibody. The solid black line represents cells stained with the LRP1 antibody. (C) Western blot for LRP1 and actin as a control for protein loading. (1) Megakaryocytes, (2) platelets, and (3) WBCs. LRP1 band is expected at approximately 85 kDa and actin at approximately 25 kDa.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Flow Cytometry, Western Blot, Derivative Assay, Staining, Labeling, Cell Culture

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: In vitro studies of the effect of RAP and anti-LRP1 antibodies on megakaryopoiesis. (A) The effect of RAP on megakaryocyte colony formation. GST indicates empty GST without conjugated RAP. Graphed is the mean percentage of megakaryocytes per well plus 1 SD. Number of experiments, each performed in duplicate, is indicated in each bar. *P = .004 versus WT cultures without PF4; **P < .003 compared with WT culture with PF4. (B) The effect of anti-LRP1 antibody (MA5A6). Ig is isoimmune control for the anti-LRP1 antibody. Mean percentage of megakaryocytes per well plus 1 SD is graphed. Number of experiments done in duplicate is indicated in each bar. *P = .004 compared with WT without PF4; **P < .003 compared with WT with PF4; ***P = .04 compared with WT without PF4.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: In Vitro

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: shRNA suppression of LRP1 and megakaryocyte colony formation. (A) Representative flow cytometry of 3T3 cells (positive control for LRP1) stably transfected with different shRNA viral vectors. The solid gray line represents cells that were unstained. The broken gray line is LRP1 expression on cells expressing the empty lentiviral vector. Solid black line shows the decrease in surface LRP1 expression after stable transfection with the LRP1 shRNA virus. (B) Same as panel A except for murine bone marrow cells after culture in media containing TPO and puromycin for 5 days. The solid gray line represents isotype control. The broken gray line is cells transfected with the negative viral vector. The solid dark line represents cells transfected with LRP1 shRNA. (C) Quantitation of change in mean fluorescence index (MFI) in murine bone marrow cells transfected with virus. Data represent mean +1 SD for 3 independent experiments. Viral titers were between 1 to 2 × 1011 viral particles/mL. (D) Effect of LRP1 shRNA on megakaryopoiesis (meg) using mPF4−/− bone marrow and hPF4High bone marrow expressed relative to megakaryopoiesis with the control empty vector. ■ is relative level of megakaryocyte seen after transfection with the empty lentiviral vector, and □ is relative level after transfection with the LRP1 shRNA vector. Data represent mean +1 SD for 4 experiments, each performed in duplicate. *P < .006 for LRP1 versus negative control for hPF4. shRNA had no effect on colony formation in mPF4−/− bone marrow. (E) Effect of LRP1 shRNA megakaryocyte colony formation in mPF4−/− bone marrow treated with exogenous PF4 (25 μg/mL). Percentage of meg colonies were normalized as in panel D. Ctl indicates control studies with empty vector. Data represent mean +1 SD. Numbers in bars represent times experiments were performed (each in duplicate). *P < .008 for LRP1 versus empty virus in the presence of PF4.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: shRNA, Flow Cytometry, Positive Control, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Fluorescence, Negative Control

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: Effect of PF4 on G1ME cells and expression of LRP1. (A) The effect of PF4 on percentage of CD42+ cells after re-expression of GATA-1 by the introduction of a GATA-1-IRES-eGFP MIGR1 retrovirus with and without 25 μg/mL PF4 in serum-free media. Results are for eGFP+-transfected cells. On the left are total CD42+ cells; middle are small, CD42+ cells; and right are large, CD42+ cells (based on forward scatter on flow cytometry). Data are shown as mean +1 SD of 4 experiments. *P < .008 comparing with and without PF4 added. (B) LRP1 expression in G1ME cells both before and after transfection with either a MIGR1 empty retrovirus (□) or MIGR1 retrovirus containing GATA-1 (♦). Figure organized as in panel A. Shown is a representative experiment of 3. (C) LRP1 expression on human cultured megakaryocytes derived from adult CD34+ bone marrow cells. Open triangles show total CD41+ cells, whereas closed triangles show LRP1+/CD41+ cells. Mean +1 SD is shown for 4 independent experiments. (D) Ploidy analysis in relation to LRP1 expression. The gray line represents cells that are CD41−. The broken line is cells that are CD41+ but LRP1−. The solid, dark line represents the cells that are positive for both LRP1 and CD41. Data are from a single experiment, but are representative of results from 5 independent experiments.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Derivative Assay